Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

Xenophagy, a type of selective autophagy, is a bactericidal membrane trafficking that targets cytosolic bacterial pathogens, but the membrane homeostatic system to cope with bacterial infection in xenophagy is not known. Here, we show that the endosomal sorting complexes required for transport (ESCRT) machinery is needed to maintain homeostasis of xenophagolysosomes damaged by a bacterial toxin, which is regulated through the TOM1L2–Rab41 pathway that recruits AAA-ATPase VPS4. We screened Rab GTPases and identified Rab41 as critical for maintaining the acidification of xenophagolysosomes. Confocal microscopy revealed that ESCRT components were recruited to the entire xenophagolysosome, and this recruitment was inhibited by intrabody expression against bacterial cytolysin, indicating that ESCRT targets xenophagolysosomes in response to a bacterial toxin. Rab41 translocates to damaged autophagic membranes via adaptor protein TOM1L2 and recruits VPS4 to complete ESCRT-mediated membrane repair in a unique GTPase-independent manner. Finally, we demonstrate that the TOM1L2–Rab41 pathway-mediated ESCRT is critical for the efficient clearance of bacteria through xenophagy.

bacteria.They identified Rab41 as a central mediator of protein recruitment to the xenophagolysossomal vacuole.Upon damage of the vacuolar membrane, through the bacterial cytolysin SLO, the increased intracytoplasmic Ca2+ concentration would trigger the recruitment of ESCRT-I, II and III machinery.In parallel, an unknown signal would promote the recruitment of TOM1L2 which in turn recruits Rab41 followed by VPS4 which interact with ESCRT-III promoting repair and limiting bacterial proliferation.This manuscript repots new findings and new concepts that are potentially interesting for researchers in the fields of cellular infection and intracellular trafficking.Besides identifying new proteins involved in the maintenance of vacuolar homeostasis, such as Rab 41 and TOM1L2, the authors claim that Rab41 involvement in this process is independent of its GTPase activity.However, in its current form the manuscript has several flaws that need to be addressed.

Major concerns:
1) The manuscript completely lacks any information on the siRNAs used.In addition, the authors do not show whether the siRNAs used are specifically down-regulating the expression of the targeted protein.The authors need to show western blots or qRT-PCR data showing the reduced expression of their targets, before taking any conclusion.
2) The authors do not used a siRNA control (such as srambelled siRNA or a siRNA targeting luciferase for example).They use as control the non-transfected cells.This is a problem, as it is not unusual that cells transfected with a control (unrelated) siRNA behave slightly different from non-transfected cells.Given the small effects shown for example in Figure 1A, where the bacterial survival goes at the best from 1 to 1.5, the use of a control siRNA is of major importance.
3) The great majority of the data is generated under overexpression of tagged proteins, including microscopy, immunoprecipitation, protein-protein interaction assays.The authors need to make the effort to corroborate their data in conditions of endogenous expression.4) Some data was obtained from the analysis of n>10 vacuoles.This is vague and might not be enough.Were those analysis done in blind?(not mentioned) To how many cells does it corresponds?5) The fact that the role of Rab41 in this process in independent of its GTPase activity is surprising.This implies that Rab41 is acting here as an adaptor protein, which is somehow a perturbing concept.Any evidence showing that the Rab41 mutants used and indeed dead for its GTPase activity?Other concerns: 1) Figure 1d shows different numbers of experiments per condition, please explain why.
2) Figure 2c is poorly convincing given the overall labelling of SNF8.Its recruitment to the GcAVs is not obvious.
3) Figure 1d in the y axis please correct CHMP4B 4) Figure 2f, g the differences are really small and the data is obtained from only 10 GcAVs.These data are not strong.Have the authors performed statistical analysis?5) From figure 3 onwards there is a problem in the text references to panel figures.Some panels do not exist and other are incorrect.This needs to be corrected, otherwise it is difficult to follow.As an example, in line 176 the panel d is not showing what is claimed.Panel d needs to be mentioned in line 178 together with panel e.Panel f which is mentioned in line 178 needs to be mentioned in line 180.Line 165 mentions a panel h which does not exist in figure 3. 6) Concerning supplementary figure 1, I was wondering if cytolysins produced by S. aureus are forming pores large enough to allow Ca2+ efflux from the vacuole?If not, what is the signal for the recruitment of CHMP4B?7) In figure 4, the major issue concerns the overexpression of proteins, which is not the best way assess protein-protein interactions.8) In figure 5a the DAPI staining clearly show dots outside the cell nucleus, which can be compatible with bacteria.Please explain.9) Figure 6d and 6e, the data is difficult to evaluate and appreciate.The authors need to show the levels of the GFP immunoprecipitated (IP GFP, IB GFP).Only then we can appreciate the levels of FLAG immunoprecipitated and concluded whether FLAG precipitates more or less in a condition that in the other.

Experimental Procedures:
The methods need to be carefully reviewed.In their current form, a lot of information and important details are missing thus preventing the validation or replication of the data by other labs.The authors need to provide detailed description of the experimental procedures they follow, if this is too extensive they can add it as supplementary material.Below follows a non-exhaustive list of issues raised by the "experimental procedures": 1) Gentamicin (50 ug/ml) is added to cells in culture.Why?At such high concentration gentamicin is able to enter into cells.In addition to eliminate extracellular bacteria during infection experiments, the concentration of gentamicin is increased to 100 ug/ml for long time periods (until 6h).This seems excessive.How can the authors ensure that gentamicin in not entering into cells and killing part of the intracellular bacteria?2) Line 440 indicates that several DNA fragments encoding human proteins were obtained by PCR on total mRNA from HeLa or HEK293T cells.The later are not human cells, they are from Hamster.This needs to be corrected.What fragments encode human and non-human proteins.
3) The sequences of all the primers and siRNAs used are missing.This needs to be added.More details on siRNAs are required: sequences, how many siRNAs per gene, methods and protocols for transfection, time between transfection and analysis, … 4) Line 485: the authors mentioned "as described previously" … where?A reference is needed or the full protocol is required.5) The authors provide a brief description of the purification method for the GST tagged protein (lines 491-495).What about the MBP-tagged protein?This protocol is missing.Please add it.6) On fluorescence microscopy the authors should provide the specifications of the camera and objectives used.In addition, they don't mention how the images have been treated?Which software used?Are the images shown projections or single plans?7) Line 555, the authors mentioned Pearson's coefficient.I couldn't find where they use it.2) Data obtained from overexpression or endogenous proteins need to be clearly indicated in the legend.
3) Abbreviations in the figure need to be full written in the legend (for example in Figure 4j, what CBB means?Coomassie brilliant Blue?Not clear.)4) The number of experiments and the N for the different panels need to be clearly mentioned.In the way they are presented is difficult to understand.For example, in figure 1d the different conditions presented in the graph do not have the same number of dots.Does this mean that the number of experiments is not the same?Also mentioning that n>x is weird, would be preferable to mention x<n<y.5) Also in Figures 1g, 2f The manuscript by Nozawa et al sheds light on the role of ESCORT machinery and Rab41/ TOMIL2 complex in the process of xenophagy.Through series of experiments carried out using a great variety of microscopic and other biochemical techniques, they provide a detailed, step-by-step description of the process.Overall, the manuscript is well-composed, and the figures are organized and well-presented.Please find my more detailed comments below.
Abstract: the abstract can benefit from inclusion of the names of techniques and details of the experimental method that generated reported findings.For example, the sentence in lines 21-24 can be edited to state: "Confocal microscopy revealed that the ESCORT components were recruited…" and so on.These additions will make the abstract more informative and improve readers' experience with it.
Quantitative methodologies and descriptive statements: there are several statements through the manuscript that can benefit from more specific quantitative comparisons.For example, in line 263 the authors report that the recruitment of Rab41 increased through the time of infection.Can you be more specific by how much?At what time point?The descriptiveness of this statement can be mitigated by the % comparison or a similar metric.Likewise, the finding in line 190 states that "these signals rarely colocalized."Can this be quantified?"Rarely" appears as a vague descriptor.Many other findings through the manuscript can be better supported by more explicit quantitative comparisons.
IP-MS experiment in Fig. 6A: I found multiple issues with the IP-MS experiment and presentation of its results.First, there appear to be no replicates.IP and AP-MS experiments are notoriously variable, and it is a standard in the field to include 3-4 replicates of each condition.The quantified protein abundances across those are then averaged and some statistical testing or software-based methodology (e.g., SAINT or COMPASS) are employed to distinguish the true interactors from the commonplace contaminants and non-specific binders.To identify interactors of Rab41, the authors have employed some unclear cutoff of FC of ~5-fold between LLOMe and DMSO treated samples.More rigorous approach and statistical testing is needed to establish which proteins are true interactors in this experiment.
Further, GFP alone control was included but not used in any way.The authors should filter out all protein identifications that appear in in experimental samples at the levels comparable to the GFP only control, as these proteins cannot be true interactors of Rab41 and are most certainly nonspecific contaminants.
Lastly, the provided heat map of the results is very misleading, as it depicts protein categories across all detected proteins in all sample types.The vast majority of these proteins are not specific to Rab41 or the treatment conditions, and having them highlighted in the figure is confusing and adds no value.
Given the extent of the follow-up experiments, TOM1L2 was likely correctly identified by the IP-MS experiments.However, the rest of the proteins that appear in the figure should be treated with caution and cannot be confidently presumed to be involved with Rab41.The authors should consider redoing the IP-MS experiment including 3-4 replicates and employing more statistically rigorous approach to interactor detection.These finding could be presented as a heat map of filtered interactors with protein classes marked, like in the current Fig 6A, or a simple table.Alternatively, the authors can perform a targeted experiment quantifying abundances of several highlighted proteins across sample types, validating their pilot data presented in Fig. 6A.
Reviewer #1 (Remarks to the Author): The manuscript of Takashi Nozawa et al. focuses on the role of Rab GTPases and ESCRT machinery in xenophagy.The authors developed a siRNA screening towards Rab GTPases and found that knockdown of Rab8A, Rab19, Rab41, and Rab44 affect GAS proliferation through ATG5-dependent autophagy.To characterize the role of Rab41 involved in GAS elimination, the authors suggested Rab41 recruits VPS4 to SLO-damaged GcAVs and facilitates the membrane repair by ESCRT complex.Rab41 localizes to damaged endomembrane independent of its GTPase activity and prenylation.They further identified TOM1L2 by Rab41 immunoprecipitation in response to endomembrane injury.Together, the authors proposed a TOM1L2-Rab41-VPS4 axis that distributes on the damaged compartments/bacteria-containing vacuoles, facilitates ESCRT-mediated membrane repair, and restricts bacterial survival.
This work provides some novel mechanistic insights into endomembrane homeostasis.
However, the overall phenotype is inconspicuous, and some conclusions are not convincing.
Therefore, the authors have to answer my comments before acceptance.
We would like to thank the reviewer for providing valuable comments regarding the significance of our study.The reviewer also raised valid and significant concerns, which have helped us tremendously in improving this revised manuscript.

Major comments:
Most of the conclusions were obtained by gene siRNA knockdown throughout the manuscript.
Due to the variable efficiency of gene knockdown, it is difficult for the authors to draw definite conclusions.For example, to demonstrate one of the most important findings that Rab41 and TOM1L2, but not ESCRT-III, are required for VPS4 localization to damaged membranes.The authors should generate Rab41 and TOM1L2 knockout cell lines (since Rab41 and TOM1L2 are not essential genes) and examine whether VPS4 is recruited to GcAVs.The data present in Fig. 4b and c are not convincing.
We thank the reviewer for constructive advice.As suggested, we have generated Rab41 and TOM1L2 knockout cell lines and investigated the involvement of these proteins in VPS4-mediated GcAV homeostasis and host defense against intracellular bacteria.Knockout of Rab41 significantly reduced the VPS4 signal on GcAVs, and complementation of Rab41 expression rescued VPS4 recruitment (Fig. 4d, e).In addition, the acidification of xenophagolysosomes and suppression of bacterial growth were also diminished by Rab41-knockout (Fig. 1h, i, j).Knockout of TOM1L2 also decreased VPS4 recruitment to xenophagolysosome and impaired bactericidal activity (Fig. 8h, i, j, k).In the revised manuscript, we concluded that Rab41 and TOM1L2 are critical for VPS4 dynamics in xenophagy.
The TOM1L2-Rab41 and Rab41-VPS4 interactions are essential parts of this manuscript.
Mutations of the GTPase activity or C-term of Rab41 did not affect VPS4 recruitment in Fig. 4, which indicates these mutations have no effect on the binding of both TOM1L2-Rab41 and Rab41-VPS4 interfaces.The authors need further characterize the critical residues of Rab41 that are required for interaction with TOM1L2 and VPS4, respectively.And then complementation of these mutants in Rab41 KO cells to validate.
As suggested by the reviewer, we have tried to identify the critical residues of Rab41 that are important for the interaction with VPS4 and TOM1L2.We successfully identified S94 in Rab41 as a critical amino acid to interact with VPS4 in the revised manuscript (Fig. 4l).We mentioned these results as "Phosphorylation of Rab GTPase is known as a possible regulatory mechanism, and the phosphorylation sites are located in conserved Rab family/Rab subfamily motif and complementarity determining regions.We next substituted candidate phosphorylation site S94 to A (Alanine) in Rab41 and found that S94A mutation reduced the interaction with endogenous VPS4 (Fig. 4l)."In addition, exogenic expression of Rab41 S94A mutant in Rab41-knockout cells did not recover bactericidal activity (Fig. 4m), suggesting that S94 residue that is critical for the interaction with VPS4 is required for Rab41-mediated xenophagy against GAS.In future studies, we will examine whether this S94 residue is phosphorylated and identify the responsible kinase, which would reveal the regulatory mechanism of xenophagy in response to bacterial infection.
In Fig. 6d and e, the Rab41-TOM1L2 interaction was not obviously enhanced upon LLOMe stimulation or GAS infection.The authors should provide immunoblot results of the interaction between Rab41 and endogenous TOM1L2 with or without LLOMe or GAS treatment.
We have examined endogenous Rab41-TOM1L2 interaction in the revised manuscript.Our coimmunoprecipitation assay demonstrated that Rab41 interacts with TOM1L2 in response to LLOMe treatment (Fig. 7d), consistent with Rab41-binding protein proteome analysis (Fig. 7a).
If I understand correctly, STAND-HS1 blocks the pore-forming activity of SLO.However, xenophagy induction also requires SLO-mediated vacuolar damage.It is impossible to distinguish the membrane damage and xenophagy induction by anti-SLO intrabody.The conclusion in Fig. 3 is suspicious.
In this experiment, STAND-HS1 is expressed in the cytosol, therefore STAND-HS1 can access cytosolic bacteria but not bacteria inside endosome (outer luminal environment).Hence, STAND-HS1 does not inhibit induction of xenophagy (damage to endosomal membranes), but only membrane damage to xenophagolysosomes (which incorporate cytoplasmic components).This is illustrated in Fig. 3i with an explanation in the revised manuscript.Does LLOMe-induced VPS4 activation also require Rab41 and TOM1L2?And please discuss the similarities and differences between LLOMe and GAS infection-caused endomembrane injury.
We thank the reviewer for constructive advice.This point is essential to identify the function of Rab41.First, we need to explain the effects of Rab41-knockdown on ESCRT-III (CHMP4B) dynamics during LLOMe-treatment.CHMP4B-positive small vacuoles appeared 10 min after LLOMe treatment and decreased after 60 min in control cells (Fig. 6a, b).By contrast, VPS4Adepleted cells harbored numerous small CHMP4B dots even after 60 min and showed large CHMP4B-positive vacuoles (Fig. 6a, b, c).Importantly, in Rab41 knockdown cells, small CHMP4B puncta reduced from 10 to 60 min after LLOMe treatment, but enlarged CHMP4B-positive vacuoles were accumulated (Fig. 6a, b, c).These enlarged vacuoles were positive for both LC3 and LAMP1, suggesting that Rab41 is mainly involved in the homeostasis of autolysosomes through VPS4 and ESCRT machinery.Indeed, VPS4 was not targeted to LC3-positive enlarged lysosomes (autolysosomes) in Rab41-knockout cells (Fig. 6e).This theory is consistent with the data of kinetics of Rab41 recruitment to lysosomes, in which endogenous Rab41 is distributed to lysosomes after LC3 and LGALS3 recruitment (Fig. 5m).Even in case of GAS infection, recruitment of Rab41 to around bacteria was abolished by ATG7-knockout, indicating that Rab41 targets xenophagic membrane.So, why should Rab41 selectively function in autolysosomal homeostasis?The reasons for this are not yet clear, but we have discussed in the Discussion section of this revised manuscript as "However, both exogenous and endogenous EmGFP-Rab41 targeted only LC3-positive bacteriacontaining vacuoles, and knockout of ATG7 abolished Rab41 recruitment to GAS.Therefore, it is likely that Rab41 functions in the homeostasis of autophagic lysosomal vacuoles.Why is Rab41 not involved in recruiting VPS4 to lysosomes and its repair but only needed for xenophagolysosomes repair?The endosomal membrane damage induces xenophagy (lysophagy), which can be eliminated, but for the membrane damage of the last-resort xenophagolysosome, another immune system should be activated to protect the cells.Of note, RNF5 and RNF185 were identified to interact with Rab41 upon lysosomal membrane damage (Fig. 7a).Because RNF5 and RNF185 have been reported to facilitate various innate immune responses to pathogens, it would be informative to reveal how Rab41 associates with these E3 ligases and their contribution to host defense systems.".
Whether Rab41 and TOM1L2 are also involved in other ESCRT-mediated functions?Such as vesicle budding and plasma membrane repair.
We have not examined the involvement of Rab41 and TOM1L2 in other ESCRT-mediated functions yet, but we are also interested in this point and have plan to investigate it in future studies.

Minor comments and errors:
The authors proposed that TOM1L2 interacts with Rab41 in response to endomembrane injury.
Is the association of Rab41 with VPS4 regulated by membrane damage or constitutive?Are VPS4 and other ESCRT proteins present in the MS results of the GFP-Rab41 interactome?
We examined endogenous Rab41-VPS4 interaction and found that Rab41 binds to VPS4 in both non-infected and infected conditions (Fig. 4g).VPS4A was also detected as Rab41-binding protein in Fig. 7A, but VPS4 was not significantly enriched in LLOMe-treated cells.These results are also consistent with the results of endogenous Rab41-VPS4 interaction.
Human has two isoforms of VPS4, VPS4A and VPS4B.The authors did not specify which VPS4 was tested.
We have examined the interaction between Rab41 and VPS4A or VPS4B, and showed that Rab41 can interact with both VPS4A and VPS4B (Fig. S6a).
As suggested, we modified "Rab41" to "Rab44" in the revised manuscript.
Line 48, does ESCRT-0 account for a complex of the ESCRT machinery?
We have modified to "ESCRT-I" in this revised manuscript.This manuscript repots new findings and new concepts that are potentially interesting for researchers in the fields of cellular infection and intracellular trafficking.Besides identifying new proteins involved in the maintenance of vacuolar homeostasis, such as Rab 41 and TOM1L2, the authors claim that Rab41 involvement in this process is independent of its GTPase activity.However, in its current form the manuscript has several flaws that need to be addressed.
We appreciate the reviewer for bringing up valid and significant concerns, which have greatly assisted us in enhancing this revised manuscript.

Major concerns:
The manuscript completely lacks any information on the siRNAs used.In addition, the authors do not show whether the siRNAs used are specifically down-regulating the expression of the targeted protein.The authors need to show western blots or qRT-PCR data showing the reduced expression of their targets, before taking any conclusion.
We have added all information of siRNAs (ID numbers) in Experimental Procedure section of this revised manuscript.We also showed the effects of siRNA knockdown on the expressions of target proteins by real-time quantitative PCR (RT-qPCR) or immunoblot (Supplementary Fig. 1a, 3e and ).
The authors do not used a siRNA control (such as srambelled siRNA or a siRNA targeting luciferase for example).They use as control the non-transfected cells.This is a problem, as it is not unusual that cells transfected with a control (unrelated) siRNA behave slightly different from non-transfected cells.Given the small effects shown for example in Figure 1A, where the bacterial survival goes at the best from 1 to 1.5, the use of a control siRNA is of major importance.
We agree with that negative control using siRNA control is critical in these experiments.In our experiment, we used non-targeting siRNA (siNT, 12935300 or 4390843, Thermo Fisher Scientific) as a negative control and quantified the effects of Rab knockdown compared to siRNA control cells.
The great majority of the data is generated under overexpression of tagged proteins, including microscopy, immunoprecipitation, protein-protein interaction assays.The authors need to make the effort to corroborate their data in conditions of endogenous expression.
We thank the reviewer for constructive advices.As suggested, we have examined endogenous protein-protein interactions (Rab41-VPS4 and Rab41-TOM1L2).Our endogenous coimmunnoprecipitation demonstrated that Rab41 interacts with both VPS4 and TOM1L2 (Fig. 4g and 7d), and also showed that Rab41-TOM1L2 interaction is triggered upon LLOMe treatment (Fig. 7d).Moreover, to observe endogenous localization of Rab41, we have generated GFP knock-in (EmGFP-Rab41) HeLa cell lines using CRISPR/Cas9 genome editing and TrueTag donor DNA (Thermo Fisher Scientific) (Supplementary Fig. 7a, b).Endogenous EmGFP-Rab41 (eEmGFP-Rab41) clearly colocalized with CHMP4B-positive bacteria (Fig. 5c) and redistributed to autolysosomal compartment in response to membrane damage (Fig. 5k, l, m).Some data was obtained from the analysis of n>10 vacuoles.This is vague and might not be enough.Were those analysis done in blind?(not mentioned) To how many cells does it corresponds?
As suggested by the reviewer, we have re-analyzed and increased n number to validate previous results.For example, in an experiment to quantify CHMP4B recruitment, we did not look at the CHMP4B signal, but took random pictures and quantify the CHMP4B and LC3 signals by image analysis.In case we analyzed 20 vacuoles, which corresponds to about 60~100 cells.
The fact that the role of Rab41 in this process in independent of its GTPase activity is surprising.This implies that Rab41 is acting here as an adaptor protein, which is somehow a perturbing concept.Any evidence showing that the Rab41 mutants used and indeed dead for its GTPase activity?Rab41 mutants used in this study are T44N and Q89L mutants of Rab41.T44 and Q89 residues are located in conserved motif among Rab GTPase proteins well-known to be important for hydrolysis of GTP to GDP and exchange from GDP to GTP, respectively.Indeed, Rab41 T44N is reported to inhibit Rab41-mediated ER-to-Golgi trafficking (PMID: 23936529), indicating that Rab41 has role in transport pathway in GTPase-dependent mechanism.We then investigated whether Rab41 mutants used in this study have GTPase activity or dead for the activity using RUSH (Retention Using Selective Hocks) assay.We observed localization of H<FEHJ<H "O%39DDEI@;9I< 11%7,5%C-?<HHM#$L?@:? @I JH9==@:A<; =HEC J?< /6 JE J?< 0EB>@ @D J?< presence of biotin, and quantified the reporter signals reached to the Golgi (GM130) after treatment of biotin for 1 h.Overexpression of wild-type and Q89L Rab41 did not affect the trafficking, but Rab41 T44N significantly reduced the transport of the reporter from the ER to the Golgi (Supplementary Fig. 4a, b), demonstrating that Rab41 T44N expression diminished GTPase-dependent function of Rab41.Whereas, expression of Rab41 T44N rescued the recruitment of VPS4 to GcAVs in Rab41 KO cells (Fig. 4d, e) and inhibited bacterial proliferation (Fig. 4f) at same content with wild-type Rab41.Therefore, it was demonstrated that Rab41 functions as an adaptor protein in GTPase-independent manner.

Other concerns:
Figure 1d shows different numbers of experiments per condition, please explain why.
This analysis was performed by several researchers in blind, and one did only one or two siRab samples.This is the reason why number of experiments differ among Rabs, however, there is no problem in statistical significance.
Figure 2c is poorly convincing given the overall labelling of SNF8.Its recruitment to the GcAVs is not obvious.
We have changed to another more representative images (Fig. 2c).

Figure 1d in the y axis please correct CHMP4B
We modified spell of CHMP4B in y axis (Fig. 2d).
Figure 2f, g the differences are really small and the data is obtained from only 10 GcAVs.These data are not strong.Have the authors performed statistical analysis?
In this revised experiment, this experiment was repeated to increase the number of GcAVs analyzed (< 30 vacuoles in each condition).We performed this experiment to understand the kinetics of the recruitment of ESCRT-III to autophagic membrane.Therefore, we did not compare the recruitment of CHMP4B and LAMP1 to LC3.
From figure 3 onwards there is a problem in the text references to panel figures.Some panels do not exist and other are incorrect.This needs to be corrected, otherwise it is difficult to follow.In figure 4, the major issue concerns the overexpression of proteins, which is not the best way assess protein-protein interactions.
We have performed endogenous protein-protein co-immunoprecipitation analysis and showed endogenous Rab41 interacts with VPS4 (Fig. 4g).
In figure 5a the DAPI staining clearly show dots outside the cell nucleus, which can be compatible with bacteria.Please explain.
These DAPI-staining dots were frequently observed when we transiently transfected plasmids.Thus, it is likely to be an aggregate of plasmids.
Figure 6d and 6e, the data is difficult to evaluate and appreciate.The authors need to show the levels of the GFP immunoprecipitated (IP GFP, IB GFP).Only then we can appreciate the levels of FLAG immunoprecipitated and concluded whether FLAG precipitates more or less in a condition that in the other.
In this revised manuscript, we have examined the interaction of endogenous Rab41 and TOM1L2 and showed that TOM1L2 was precipitated with endogenous Rab41 only when cells were treated with LLOMe (Fig. 7d).

Experimental Procedures:
The methods need to be carefully reviewed.In their current form, a lot of information and important details are missing thus preventing the validation or replication of the data by other labs.The authors need to provide detailed description of the experimental procedures they follow, if this is too extensive they can add it as supplementary material.Below follows a nonexhaustive list of issues raised by the "experimental procedures": We thank for your suggestion.We have fully revised Experimental Procedure Section in the revised manuscript.
Gentamicin (50 ug/ml) is added to cells in culture.Why?At such high concentration gentamicin is able to enter into cells.In addition to eliminate extracellular bacteria during infection experiments, the concentration of gentamicin is increased to 100 ug/ml for long time periods (until 6h).This seems excessive.How can the authors ensure that gentamicin in not entering into cells and killing part of the intracellular bacteria?Cell lines are frequently maintained medium with streptomycin and penicillin to inhibit microbial contaminations, but GAS JRS4 strain used in this study is resistant to streptomycin.
We then used gentamicin instead of streptomycin and penicillin.As you point out, it has been noted that some cell types and treatment concentrations can penetrate into cells, so it is important to set up carefully according to the experimental conditions (PMID: 29312891).The concentrations of gentamicin used in this study are frequently (in more than hundreds of papers) used in "Gentamicin protection assay" to completely kill the extracellular bacteria in HeLa cells.
GAS strains including JRS4 used in this study is sensitive to gentamicin, but the number of survival bacteria increase with time in HeLa cells ( Hamster.This needs to be corrected.What fragments encode human and non-human proteins.
As suggested, we separately described the mRNA from HeLa or HEK293T.HEK293T is a cell line of human embryonic kidney.
The sequences of all the primers and siRNAs used are missing.This needs to be added.More details on siRNAs are required: sequences, how many siRNAs per gene, methods and protocols for transfection, time between transfection and analysis, … We have added siRNA information in Experimental procedure in this revised manuscript.We also revised as follow " For knockdown experiments, HeLa cells were seeded at 4 x 10 4 cell/well in 24-well plate and 10 pmol siRNA oligonucleotides Rab1A (s229381; Thermo Fisher Scientific), Rab1B (s119; Thermo Fisher Scientific), Rab2A (s11660; Thermo Fisher Scientific), Rab2B (s39689; Thermo Fisher Scientific), Rab3A (s11668; Thermo Fisher Figure legends: In general, the Figure legends are poorly written.They need to be improved to provide more details on what they show.As a few examples: 1) They should inform whether confocal microscopy images are full projections of different plans or instead they are single plans.2) Data obtained from overexpression or endogenous proteins need to be clearly indicated in the legend.3)Abbreviations in the figure need to be full written in the legend (for example in Figure4j, what CBB means?Coomassie brilliant Blue?Not clear.)4) The number of experiments and the N for the different panels need to be clearly mentioned.In the way they are presented is difficult to understand.For example, in figure1dthe different conditions presented in the graph do not have the same number of dots.Does this mean that the number of experiments is not the same?Also mentioning that n>x is weird, would be preferable to mention x<n<y.5) Also in Figures1g, 2f, 2j,5b the authors need to indicate to what corresponds the shadow lines.Is that data dispersion?Need clarification.
Figure legends: In general, the Figure legends are poorly written.They need to be improved to provide more details on what they show.As a few examples: 1) They should inform whether confocal microscopy images are full projections of different plans or instead they are single plans.2) Data obtained from overexpression or endogenous proteins need to be clearly indicated in the legend.3)Abbreviations in the figure need to be full written in the legend (for example in Figure4j, what CBB means?Coomassie brilliant Blue?Not clear.)4) The number of experiments and the N for the different panels need to be clearly mentioned.In the way they are presented is difficult to understand.For example, in figure1dthe different conditions presented in the graph do not have the same number of dots.Does this mean that the number of experiments is not the same?Also mentioning that n>x is weird, would be preferable to mention x<n<y.5) Also in Figures1g, 2f, 2j,5b the authors need to indicate to what corresponds the shadow lines.Is that data dispersion?Need clarification.
As an example, in line 176 the panel d is not showing what is claimed.Panel d needs to be mentioned in line 178 together with panel e.Panel f which is mentioned in line 178 needs to be mentioned in line 180.Line 165 mentions a panel h which does not exist in figure 3. We thank the reviewer for suggestions.We have carefully modified the panel figures and the citation in the revised manuscript.Concerning supplementary figure 1, I was wondering if cytolysins produced by S. aureus are forming pores large enough to allow Ca2+ efflux from the vacuole?If not, what is the signal for the recruitment of CHMP4B? S. aureus produces cytolysins such as a-toxin and to form pores large enough to induce calcium mobilization.
Fig. A).Moreover, although knockout of ATG5 increased intracellular bacteria (Fig. A), increase of gentamicin concentration to 200 ug/ml did not change the survival bacteria at all (Fig. B).Therefore, of course we cannot exclude the possibility that small part of intracellular bacteria are killed by gentamicin, but we do not believe that this concentration of gentamicin has an effect sufficient to overturn the experimental results.Line 440 indicates that several DNA fragments encoding human proteins were obtained by PCR on total mRNA from HeLa or HEK293T cells.The later are not human cells, they are from